Application of four genotyping methods to Mycoplasma bovis isolates derived from western Canadian feedlot cattle

Authors: KINNEAR, ANDREA - UNIVERSITY OF SASKATCHEWAN; WALDNER, MATTHEW - UNIVERSITY OF SASKATCHEWAN; MCCALLISTER, TIM - LETHBRIDGE RESEARCH CENTER; ZAHEER, RAHAT - LETHBRIDGE RESEARCH CENTER; REGISTER, KAREN; JELINSKI, MURRAY - UNIVERSITY OF SASKATCHEWAN

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/2021
Publication Date: 6/18/2021
Citation: Kinnear, A., Waldner, M., Mccallister, T., Zaheer, R., Register, K.B., Jelinski, M. 2021. Application of four genotyping methods to Mycoplasma bovis isolates derived from western Canadian feedlot cattle. Journal of Clinical Microbiology. 59(7). Article e0004421. https://doi.org/10.1128/JCM.00044-21.
DOI: https://doi.org/10.1128/JCM.00044-21

Interpretive Summary: Mycoplasma bovis is a costly pathogen affecting cattle production and health around the world. Understanding the epidemiology of mycoplasmosis and investigating possible connections between particular genetic types and clinical disease presentation requires reproducible, standardarized and highly discriminatory typing methods. This study compares four methods for molecular typing of M. bovis isolates, each of which uses data obtained from genome sequencing: 1) multilocus sequence typing (MLST), 2) core genome MLST (cgMLST), 3) core genome single nucleotide variant (cgSNV) analysis, and 4) whole genome single nucleotide variant (wgSNV) analysis. Each method was used to evaluate 129 isolates obtained from the nasal cavity, lung or joint of cattle in Canadian feedlots obtained between 2006 and 2018. There was good concordance among all four methods but some methods require higher quality genome sequence data than others. wgSNV performed best since it was able to distinguish the largest number of strains. However, the software needed for wgSNV requires specialized training that may not be widely available. Overall, each method has a place in strain typing contingent on the research question being addressed. The choice of which method is best for the laboratory conducting typing analysis depends on the quality of the data available and the availability of specialized software and training.

Technical Abstract: Four different methods of phylogenetic analysis were compared using Mycoplasma bovis isolates (n equals 129) derived from feedlot cattle of varying health status (healthy, diseased, dead), from different anatomical locations (nasopharynx, lung, joint), and over a 12 year period (2006-2018). Four in-silico typing methods were applied using whole genome sequencing (WGS) data: multilocus sequence typing (MLST), core genome MLST (cgMLST), core genome single nucleotide variant (cgSNV) analysis, and whole genome single nucleotide variant (wgSNV) analysis. Overall, there was good concordance amongst the four methods, with MLST having the lowest resolution, while the wgSNV analysis provided the greatest resolution. The two cg methods yielded extremely similar clade structures, with a resolution that was marginally less than that of wgSNV. The MLST analysis was found to be robust, easy to apply, and the extensive PubMLST M. bovis database facilitated comparison of STs derived from other unrelated studies. Conversely, wgSNV was the least reliant upon the quality of the sequencing data, and provided the higest degree of resolution. However, the software applications require specialized training for running the analysis. In this study, the resolution of the cg methods was very good, and the graphical interface software (Ridom SeqSphere+) was intuitive and relatively user-friendly, allowing non-bioinformaticians with a moderate level of relevant background knowledge to analyze the data. A secondary objective was to determine if one or more methods could relate genotypes to phenotypes (host’s health status, anatomical location, year, and feedlot of origin); no associations were found.